IJMS | Free Full-Text | Different miRNAs Related to FBXW7 Mutations or High Mitotic Indices Contribute to Rectal Neuroendocrine Tumors: A Pilot Study

1. Introduction

Neuroendocrine tumors (NETs) are a heterogeneous group of tumors originating from neuroendocrine cells and harbor malignant potential [1,2]. The incidence of NETs is becoming more common worldwide, and their characteristics and behavior can vary widely [1,2]. The incidence of gastrointestinal NETs depends on the primary tumor site, with the greatest prevalence in the rectum [3,4,5]. Rectal NETs are the most frequent in Asia, including the Republic of Korea, taking up 60–89% of all gastroenteropancreatic NETs [3,4,5]. In particular, a nine-fold increase in incidence has been discovered in the Republic of Korea [5]. Most rectal NETs are small (<10 mm) and have a favorable 5-year overall survival rate of 88% [6]. However, its prognostic implications are exceedingly discussed because the tumor behavior varies according to the grade, size, invasion depth, and lymphovascular invasion (LVI) [7,8,9,10,11,12,13,14]. Moreover, its incidence may reflect ethnic or geographical differences, as rectal NETs develop predominantly in Asians. Midgut NETs, including the small intestine, occur more in white and less in Asian patients [7,8,9,10,11,12,13,14]. Virtually, the genetic mutations observed in NETs diverge depending on their anatomic location [15]. Pancreatic NETs have been illustrated to carry somatic mutations in MEN1, DAXX, ATRX, PTEN, and members of the mTOR signaling pathway [16], whereas gastrointestinal NETs have been reported to harbor CDKN1B mutations [15,17]. The inactivation of RB1 and TP53 has been described in so-called neuroendocrine carcinomas rather than NETs [16]. However, this genetic information is exclusively based on the European population [15,16]. Our previous study involving 69 rectal cases from single institutional samples in Korea demonstrated that one-third of well-differentiated, grade 1 or 2 rectal NETs, frequently carry TP53 (10.1%) or FBXW7 (7.2%) mutations [18]. This high prevalence of TP53 mutations differs from the European population-based molecular results [18]. There has been scarce information on the molecular profiling of rectal NETs in the Korean population, wherein piecemeal molecular characterization may limit the explanation of the spectrum of behavior in rectal NETs [18]. Therefore, there is an inevitable need to identify key regulators that could influence the distinct molecular features of rectal NETs in the Korean population.
Epigenetic regulation of gene expression, particularly through miRNA, is an important research topic linked to cancer development [19]. miRNAs are small noncoding RNA molecules that regulate gene expression at the post-translational level by suppressing target mRNAs [20]. Gene silencing by miRNAs governs diverse physiological and pathological steps, which can be used in various cancer classifications as informative tissue markers [20]. However, studies on miRNA expression profiles in NETs have yielded conflicting results [21,22,23,24], making it challenging to identify key tissue markers [25]. For example, a previous study discovered that miR-885-5p could be an early prognostic indicator for LVI in rectal NETs [21]. However, this study did not investigate the connection between miR-885-5p and tumor grade. A more recent study in Japan examined seven rectal NET tissues and found overexpression of the miR-144/miR-451 cluster in NETs of grade 1 with LVI [22]. In another study, higher expression of miR-19a and miR-96 was found in metastatic tissue than in primary tumors of the small intestine and colon; however, the sample size was limited [23]. A meta-analysis of 22 studies found inconsistent miRNA signatures in gastric, pancreatic, small intestinal, and colorectal NETs [24]. miR-222 was associated with regulated p27KIP1 in gastric NETs, while miR-21 and miR-144 showed variable up- and down-regulation in pancreatic NETs depending on the analysis method [24]. No consistent miRNA signatures were identified in small intestinal or colorectal NETs [24]. Nevertheless, a lack of data on rectal NETs requires further validation of rectal NETs in the Korean population. In this study, the Nanostring nCounter Human v3 miRNA Expression assay screens 800 miRNAs, representing one of the vast miRNA assays as high-throughput techniques, out of date.

Therefore, we screened differentially expressed miRNAs clinically or pathologically relevant to the specific genetic mutations involved in rectal NETs based on the tumor grade to identify miRNA biomarkers related to prognosis and tumor behavior.

3. Discussion

In this study, screening 584 miRNA expressions in rectal NETs revealed that FBXW7 and mitotic index were the sole candidate genes and clinicopathological features specifically relevant to miRNAs, respectively. FBXW7 acts as a potent tumor suppressor that may control the expression levels of multiple oncoproteins (i.e., mTOR, notch, cyclin E, c-Myc, and c-Jun) that engage in cellular signaling pathways by inducing them for ubiquitin-mediated proteasomal degradation, thereby protecting against cancer development [27,28]. Several studies have demonstrated that FBXW7 also significantly influences DNA damage response and repair processes, prime molecular mechanisms for maintaining cellular homeostasis, and genomic integrity [29,30,31,32]. Accordingly, FBXW7 dysregulation in this process may be a critical factor contributing to tumorigenesis [27,28]. A recent study has reported a high frequency of P53 (61%), APC (53%), FBXW7 (25%), and KRAS (25%) mutations in rectal NETs [15]. Likewise, in our previous study, FBXW7 (p.R465H) was found to be likely pathogenic in low-grade rectal NETs [18]. In this context, it may be important to focus on the epigenetic regulation of FBXW7 in rectal NETs, as FBXW7 is a critical regulator of several key signaling pathways involved in cell growth and proliferation [27,28]. Understanding the epigenetic regulation of FBXW7 in rectal NETs may provide insights into the mechanisms underlying tumor development and potentially identify novel therapeutic targets for treating these tumors.
Alterations in FBXW7 expression have been linked to the development and progression of several types of cancer [27,28]. Some FBXW7 regulators include miRNAs, such as miR-27 in lung cancer, miR-32 in breast cancer, miR-92a in cervical cancer, miR-223 in gastric cancer, miR-182 in lung cancer, and miR-223 in T-cell lymphoma [33,34,35]. In the rectal NETs of the present study, seven miRNAs (hsa-miR-769-5p, hsa-miR-221-3p, hsa-miR-34a-5p, hsa-miR-181c-5p, hsa-miR-1246, hsa-miR-324-5p, and hsa-miR-361-3p) were significantly down-regulated in FBXW7-mutant tumors. In gastric cancer, miR-223 acted as an oncogene and negatively modulated FBXW7 expression, governing proliferation, apoptosis, and invasion [33]. Consistent with this, Notch-mediated activation of miR-223 represses FBXW7 in T-cell acute lymphoblastic leukemia [34]. In breast cancer cells, miR-32 induces cell proliferation and migration, evading apoptosis by down-regulating FBXW7 [35]. In cervical cancer cells, an up-regulation of miR-92a causes a down-regulation of FBXW7, thereby stimulating tumor cell proliferation and invasion [36]. miR-182 increases the proliferation of non-small cell lung cancer cells by inhibiting FBXW7 [37].
Here, we noted that hsa-miR-769-5p was most correlated with FBXW7 mutation, further linked to ECM-receptor interaction and lysine degradation via the KEGG analysis of target signaling pathways. The ECM is a central element of both normal and tumor tissues, consisting of a complex texture of cross-linked proteins to play a role in resident cells’ hydration, elasticity, and structural organization [38]. It importantly helps in neoplastic proliferation, angiogenesis, progression, and tumor survival [39]. Specific ECM alterations regulating tissue biochemical and biomechanical properties have been described in NETs [40], which may imply that several ECM glycoproteins may contribute to the tumorigenesis of NETs. In a recent study, the up-regulation of six fibrogenic genes (COL5A2, COL1A2, COL3A1, ITGA5, ITGB1, and ITGB1) in pulmonary neuroendocrine carcinoma and the down-regulation of pulmonary neuroendocrine neoplasms were strongly related to no metastasis and overall survival [41]. Only one study has shown that the activation of the lysine degradation pathway injures tumor cell proliferation [42], which is also related to miR-769-5p tumor suppression, as shown in subsequent studies. miR-769-5p/miR-769-3p-mediated inhibition of proliferation causes apoptosis in gastric cancer cells [43]. Another study has shown that miR-769-5p inhibited non-small cell lung cancer proliferation and invasion, indicating that this type of tumor could benefit from miR-769-5p as a diagnostic and prognostic biomarker [44]. hsa-miR-769-5p has also been described to be expressed at low levels in papillary thyroid carcinomas, and the overexpression of hsa-miR-769-5p highly affected the formation of the tumor microenvironment [45], which appears to be consistent with our findings.

Up-regulated miR-3934-5p and down-regulated miR-4286, miR-223-3p, miR-129-5p, and miR-153-3p were characterized in tumors with high mitotic indices, in which miR-3934-5p was the only one statistically significant after adjustment. Further clustering analysis separated two main groups: (1) high mitotic index, grade 2, wild-type FBXW7, and wild-type TP53 and (2) low mitotic index, grade 1, FBXW7 mutation, and TP53 mutation. This may indicate that these miRNA contributions were mutually exclusive between the high mitotic index and FBXW7 mutation in rectal NETs. The KEGG pathway maps also confirmed the different pathway involvement in different miRNAs between FBXW7-mutated and highly mitotic-active tumors. hsa-miR-3934-5p may be closely implicated in a high mitotic index, eventually contributing to higher grade 2 tumors in rectal NETs.

In contrast, hsa-miR-769-5p related to FBXW7 mutation may be involved in grade 1 tumors and is less likely to be involved in tumor aggressiveness. Indeed, a recent study has discovered that WHO grade 3 gastro-pancreatic NETs tend to harbor NETs with lower FBXW7 mutation frequency than grade 1 and grade 2 NETs [15]. Comparisons of normal pancreatic islets with their matched neoplastic groups disclosed changes in ECM compositions, which also differed depending on histological grade [40,46]. This may explain the finding that the ECM-receptor interaction KEGG pathway (hsa-miR-769-5p) was significantly associated with FBXW7 mutations and was more likely involved in grade 1 tumors. hsa-miR-769-5p appears to be a possible regulator of FBXW7-mutated rectal NETs. These differentially expressed miRNAs could be potential tissue biomarkers for aggressive behavior in rectal NETs.
Our study was limited by a relatively small number of rectal NETs. We did not perform the validation step using quantitative PCR. However, we utilized the advantages of Nanostring technology to identify 24 cancer-related miRNAs out of 584 miRNAs without requiring the amplification of transcripts. We believe that our approach was able to identify low-abundance transcripts that may have been difficult to validate through PCR [47]. Our study focused on identifying candidate miRNAs. In addition, we did not analyze the difference between plasma and tissue miRNAs. miRNAs from tumors may be secreted within membrane vesicles (exosomes) or directly into the blood, indicating that miRNAs play a key role in intercellular communication [48]. Plasma miRNAs communicate the most with the pericardial, adipose, liver, and spleen tissues [49]. As rectal NETs are submucosal tumors, tissue-based analysis of our miRNAs may be more appropriate as they reflect the origin of these tumors. Because some may metastasize, we aim to further investigate the differences in plasma miRNAs between localized rectal NETs and those with metastasis to advance our understanding of these tumors in future studies.

Nevertheless, some conclusions emerged from our results. To our knowledge, this is the first study to identify a correlation between FBXW7 mutations and miRNAs in rectal NETs. hsa-miR-769-5p was most correlated with FBXW7 mutation and further linked to ECM-receptor interaction and lysine degradation via a KEGG analysis of target signaling pathways. hsa-miR-3934-5p may be strongly implicated in a high mitotic index, which may eventually contribute to higher grade 2 tumors in rectal NETs, whereas hsa-miR-769-5p related to FBXW7 mutation may be involved in grade 1 tumors and is likely to play a role in suppressing rectal NET progression.

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